Project Summary Hearing and balance are amazing senses that allow us to communicate with each other and navigate the environment. Our long-term research goals are to enhance our understanding of the molecular basis for the development and maintenance of sensory hair cells located in the inner ear. More specifically, we focus on the hair bundle, which acts as a key sensory organelle on the apical surface of all hair cells. The hair bundle is a specialized structure composed of groups of rod-shaped cellular protrusions called stereocilia. Each stereocilium is covered by the apical plasma membrane and supported internally by a tight arrangement of uniformly oriented parallel actin filaments. This proposal addresses a critical yet relatively unexplored part of the hair bundle. We will focus on the base of the hair bundle, where the shafts of stereocilia morphologically transition from a cylindrical to a conical taper shape that is confluent with the apical cytoplasm of the hair cell, and where a subset of actin filaments penetrates to the underlying actin-rich meshwork of the cuticular plate. The tapered base of stereocilia is a special compartment that acts as a pivot point for their deflection during mechanical stimulation, and helps maintain their continuous function during incessant stimulation arising from sounds and body movement. The work in this proposal aims to understand the roles of certain deafness- associated proteins that are concentrated at the base of stereocilia. We hypothesize that these proteins form an interdependent complex that connects actin filaments to the plasma membrane, specifically at the tapered base of individual stereocilia and the apical plasma membrane between stereocilia. Mutations that disrupt proteins in this complex presumably cause the apical plasma membrane to detach from underlying actin filaments, resulting in destruction of the hair bundle, loss of sensory function, and deafness. The main objectives of this project are to establish interactions between genes encoding key proteins of the complex, determine factors that regulate the distribution of the complex, and identify specific protein-protein interactions that mediate membrane-cytoskeletal attachment at the base of the hair bundle. This work will provide new insights into our understanding of the relationships among DFNB103 (Clic5), DFNB24 (Rdx), DFNA22/DFNB37 (Myo6), and DFNB79 (Ptprq) in sensory hair cells. Ultimately, this knowledge will fill gaps in our understanding of the development and maintenance of the hair bundle.